(a) Field of the Invention
The invention relates to an in vitro test by recombinant DNA technology to be able to evaluate the potency of various angiotensin II (AII) antagonists on the inhibition of expression of angiotensinogen (ANG) gene in vivo.
(b) Description of Prior Art
Angiotensin II (AII) is derived from the precursor glycoprotein, angiotensinogen (ANG) which is synthesized predominantly in the liver via successive cleavage by renin and angiotensin-converting enzyme (ACE). The evidence for a direct relationship between renin-angiotensin-system (RAS) and the development of hypertension was reported and it was demonstrated that transgenic mice which carry an exogenous angiotensinogen (ANG) gene and/or renin gene and express high levels of plasma angiotensinogen and Angiotensin II will develop high blood pressure. These studies demonstrated unequivocally that angiotensinogen is an important component for the development of high blood pressure but the expression of renin and angiotensin-converting enzyme is also equally important.
The expression of the angiotensinogen gene in mouse hepatoma (Hepa 1-6) cells was previously reported and shown that dexamethasone (DEX) stimulates the expression of fusion genes containing the 5'-flanking region of the rat angiotensinogen (ANG) gene fused with a bacterial chloramphenicol acetyl transferase (CAT) coding sequence as reporter, pOCAT (ANG N-1498/+18), in a dose-dependent manner (Ming M. et al., Am. J. Hypertens., 1993, 6:141-148). Furthermore, it was demonstrated that the addition of 8-Bromo-cAMP (8-Br-cAMP) enhanced the stimulatory effect of dexmethasone (DEX) on the expression of ANG-CAT fusion genes (Ming M. et al., Am. J. Hypertens., 1993, 6:141-148). The addition of 8-Br-cAMP alone, however, had no stimulatory effect on the expression of the ANG-CAT fusion genes. These studies suggest that DEX and cAMP might act synergistically or co-operatively to stimulate the expression of the ANG gene in the liver.
Angiotensin II (AII) is known to stimulate the synthesis and secretion of ANG in vivo and in vitro. The stimulatory effect of angiotensin II is apparently mediated via the angiotensin II-receptors (AT.sub.1 -receptors).
It would be highly desirable to be provided with means to determine if the addition of angiotensin II-receptors (AT.sub.1 - and AT2-receptors) antagonists could inhibit the stimulatory effect of angiotensin II on the expression of ANG gene.
There is disclosed in U.S. Pat. No. 5,112,757 issued in the name of Guguen-Guillouzo et al. on May 12, 1992, a method for culturing human hepatocytes so that they maintain hepatocyte functions at a high level for an extended period of time.
There is disclosed in U.S. patent application No. 07/284,368 filed on Dec. 14, 1988 in the name of Cole et al., a human liver epithelial cell line with extended life spans which more reliably predict the affect of various chemical assaults on the cell in carcinogen and mutagen research, metabolism research and other applications for cell lines.
None of these documents provides a culture of human hepatocytes which could be used to determine if the addition of angiotensin II-receptors (AT.sub.1 - and AT.sub.2 -receptors) antagonists could inhibit the stimulatory effect of angiotensin II on the expression of ANG gene.